The goal of this project is the development and application of novel multiplex quantitative real time PCR (MqrtPCR) assays for differential diagnosis inpatients who may be multiply infected with dengue and other hemorrhagic fever pathogens. The rapid and accurate diagnosis of the several possible hemorrhagic viruses in patients suspected of dengue infection is critical for the identification of a large cohort of patients whose peripheral blood sample will be examined in this program to assess the epitope-based dengue vaccine that is the primary goal of this multi-project program. These assays will also be beneficial in furthering the epidemiological surveillance of dengue infections in Brazil. A principal and unique feature of the proposed assay is its multiplex quantitative real-time PCR technology and its capacity for genotype-specific differential diagnosis of infectious agents currently endemic in Brazil: dengue viruses, West Nile virus, yellow fever virus, hantavirus and leptospira species. A multiplex assay that will simultaneously detect conserved sequences of these infectious agents, all of which may have similar clinical symptoms, will allow the rapid diagnoses of patients at an early phase of infection, an important aspect in differential diagnosis. This assay technology will also be used in multiplex format to distinguish any combination of the four dengue serotype viruses in a single operation. This assay technology has already been developed for the quantitative real time PCR assay of replicating dengue serotype 2. The optimal conditions and parameters of these assays, with respect to their sensitivity and specificity for multiplex differential diagnosis of other viruses and the four dengue serotypes will be determined in laboratory control studies. The validated protocols and reagents will then be made available to workers in Brazil for application to the field studies with patient cohorts. Accurate diagnoses of these patients are crucial if the blood samples are to be used for the ex vivo biological validation of the predicted peptide epitopes and response to the vaccine formulation described in Projects 1, 2 and 4.